Many tumor cells contain abnormal chromosome content (aneuploidy), which results in altered expression of thousands of proteins. A subset of these proteins normally form larger oligomeric assemblies with partner proteins and are unstable as ?orphan? proteins outside of these complexes. The accumulation of mutations and chromosomal abnormalities during tumor cell evolution generates an increasingly unstable proteome that elevates the need for protein quality control systems to selectively remove these unstable defective proteins. Despite the prevalence of proteins that demonstrate collaborative stability, the quality control mechanisms that govern collaborative stability and facilitate the degradation of orphan proteins is almost entirely uncharacterized in mammals. Our objective is to determine the mechanisms and cellular factors that regulate collaborative protein stability and utilize these pathways to selectively target tumors with unstable proteomes. We have identified Huwe1 as a ubiquitin ligase that targets unassembled orphan proteins for degradation. Our hypothesis is that Huwe1 is a critical cellular factor the mediates orphan protein destruction and enhances fitness in aneuploid cells with unstable proteomes. To probe this hypothesis, we will: (1) perform structure function analysis on Huwe1 to determine the mechanism of how Huwe1 selects and targets substrates; (2) identify Huwe1 substrates in normal and aneuploid cells; (3) determine the impact of loss or gain of Huwe1 function on protein homeostasis and the response to proteotoxic stress in euploid and aneuploid cells. We have identified orphan proteins that are destroyed in both a Huwe1-dependent and independent manner. We will utilize optical reporter systems based on identified substrates that rely on collaborative stability to perform CRISPR/Cas9-based genetic screens to identify unknown factors that participate in both Huwe1-dependent and independent degradation of unassembled orphan proteins. Research outcomes within this proposal will establish if targeting orphan degradation pathways represents an effective strategy to reduce fitness in tumor cells with unstable proteomes. If successful, we will establish the mechanism Huwe1 utilizes to specifically target incorporated orphan proteins for degradation and determine if aneuploid cells with unstable proteomes are sensitive to defects in orphan protein degradation pathways. We will also genetically define the orphan protein degradation pathway in mammals. The long-term goal is to develop molecular strategies aimed at reducing fitness in cells with limited protein homeostasis capacity by utilizing newly characterized quality control mechanisms identified during this research to inhibit defective orphan protein destruction. The findings resulting from the proposed research will provide substantial progress toward our goal of identifying cellular mechanisms that regulate protein quality control that can be leveraged to combat aging associated human pathology.